Throughout this specification, including any claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.
It must be noted that, as used in the specification and any appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.
Ranges are often expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment.
DNA in eukaryotic cells is tightly complexed with proteins (histones) to form chromatin. Histones are small, positively charged proteins which are rich in basic amino acids (positively charged at physiological pH), which contact the phosphate groups (negatively charged at physiological pH) of DNA. There are five main classes of histones, H1, H2A, H2B, H3, and H4. The amino acid sequences of histones H2A, H2B, H3, and H4 show remarkable conservation between species, whereas H1 varies somewhat, and in some cases is replaced by another histone, e.g., H5. Four pairs of each of H2A, H2B, H3, and H4 together form a disk-shaped octomeric protein core, around which DNA (about 140 base pairs) is wound to form a nucleosome. Individual nucleosomes are connected by short stretches of linker DNA associated with another histone molecule (e.g., H1, or in certain cases, H5) to form a structure resembling a beaded string, which is itself arranged in a helical stack, known as a solenoid.
The majority of histones are synthesised during the S phase of the cell cycle, and newly synthesised histones quickly enter the nucleus to become associated with DNA. Within minutes of its synthesis, new DNA becomes associated with histones in nucleosomal structures.
A small fraction of histones, more specifically, the amino side chains thereof, are enzymatically modified by post-translational addition of methyl, acetyl, or phosphate groups, neutralising the positive charge of the side chain, or converting it to a negative charge. For example, lysine and arginine groups may be methylated, lysine groups may be acetylated, and serine groups may be phosphorylated. For lysine, the —(CH2)4—NH2 sidechain may be acetylated, for example by an acetyltransferase enzyme, to give the amide —(CH2)4—NHC(═O)CH3. Methylation, acetylation, and phosphorylation of amino termini of histones which extend from the nucleosomal core affects chromatin structure and gene expression. (See, for example, Spencer and Davie, 1999).
Acetylation and deacetylation of histones Is associated with transcriptional events leading to cell proliferation and/or differentiation. Regulation of the function of transcription factors is also mediated through acetylafton. Recent reviews of histone deacetylation include Kouzarides, 1999 and Pazin et al., 1997.
The correlation between the acetylation status of histones and the transcription of genes has been known for over 30 years (see, for example, Howe et al., 1999). Certain enzymes, specifically acetylases (e.g., histone acetyltransferase, HAT) and deacetylases (e.g., histone deacetylase, HDAC), which regulate the acetylation state of histones have been identified in many organisms and have been implicated in the regulation of numerous genes, confirming the link between acetylation and transcription. See, for example, Davie, 1998. In general, histone acetylation correlates with transcriptional activation, whereas histone deacetylation is associated with gene repression.
A growing number of histone deacetylases (HDACs) have been identified (see, for example, Ng and Bird, 2000). The first deacetylase, HDAC1, was identified in 1996 (see, for example, Taunton et al., 1996). Subsequently, two other nuclear mammalian deacetylases were found, HDAC2 and HDAC3 (see, for example, Yang et al., 1996, 1997, and Emiliani et al., 1998). See also, Grozinger et al., 1999; Kao et al., 2000; and Van den Wyngaert et al., 2000.
Eleven (11) human HDACs have been cloned so far:                HDAC1 (Genbank Accession No. NP—004955)        HDAC2 (Genbank Accession No. NP—001518)        HDAC3 (Genbank Accession No. O15379)        HDAC4 (Genbank Accession No. AAD29046)        HDAC5 (Genbank Accession No. NP—005465)        HDAC6 (Genbank Accession No. NP—006035)        HDAC7 (Genbank Accession No. AAF63491)        HDAC8 (Genbank Accession No. AAF73428)        HDAC9 (Genbank Accession No. AAK66821)        HDAC10 (Genbank Accession No. AAK84023)        HDAC11 (Genbank Accession No. NM—024827        
These eleven human HDACs fall in two distinct classes: HDACs 1, 2, 3 and 8 are in class I, and HDACs 4, 5, 6, 7, 9, 10 and 11 are in class II.
There are a number of histone deacetylases in yeast, including the following:                RPD3 (Genbank Accession No. NP—014069)        HDA1 (Genbank Accession No. P53973)        HOS1 (Genbank Accession No. Q12214)        HOS2 (Genbank Accession No. P53096)        HOS3 (Genbank Accession No. Q02959)        
There are also numerous plant deacetylases, for example, HD2, in Zea mays (Genbank Accession No. AF254073—1).
HDACs function as part of large multiprotein complexes, which are tethered to the promoter and repress transcription. Well characterised transcriptional repressors such as Mad (Laherty et al., 1997), pRb (Brehm et al., 1998), nuclear receptors (Wong et al., 1998) and YY1 (Yang et al., 1997) associate with HDAC complexes to exert their repressor function.
The study of inhibitors of histone deacetylases indicates that these enzymes play an important role in cell proliferation and differentiation. The inhibitor Trichostatin A (TSA) (Yoshida et al., 1990a) causes cell cycle arrest at both G1 and G2 phases (Yoshida and Beppu, 1988), reverts the transformed phenotype of different cell lines, and induces differentiation of Friend leukaemia cells and others (Yoshida et al., 1990b). TSA (and SAHA) have been reported to inhibit cell growth, induce terminal differentiation, and prevent the formation of tumours in mice (Finnin et al., 1999).

Cell cycle arrest by TSA correlates with an increased expression of gelsolin (Hoshikawa et al., 1994), an actin regulatory protein that is down regulated in malignant breast cancer (Mielnicki et al., 1999). Similar effects on cell cycle and differentiation have been observed with a number of deacetylase inhibitors (Kim et al., 1999).
Trichostatin A has also been reported to be useful in the treatment of fibrosis, e.g., liver fibrosis and liver cirrhosis. See, e.g., Geerts et al., 1998.
Recently, certain compounds that induce differentiation have-been reported to inhibit histone deacetylases. Several experimental antitumour compounds, such as trichostatin A (TSA), trapoxin, suberoylanilide hydroxamic acid (SAHA), and phenylbutyrate have been reported to act, at least in part, by inhibiting histone deacetylase (see, e.g., Yoshida et al., 1990; Richon et al., 1998; Kijima et al., 1993). Additionally, diallyl sulfide and related molecules (see, e.g., Lea et al., 1999), oxamflatin (see, e.g., Kim et al., 1999; Sonoda et al., 1996), MS-27-275, a synthetic benzamide derivative (see, e.g., Saito et al., 1999; Suzuki et al., 1999; note that MS-27-275 was later re-named as MS-275), butyrate derivatives (see, e.g., Lea and Tulsyan, 1995), FR901228 (see, e.g., Nokajima et al., 1998), depudecin (see, e.g., Kwon et al., 1998), and m-carboxycinnamic acid bishydroxamide (see, e.g., Richon et al., 1998) have been reported to inhibit histone deacetylases. In vitro, some of these compounds are reported to inhibit the growth of fibroblast cells by causing cell cycle arrest in the G1 and G2 phases, and can lead to the terminal differentiation and loss of transforming potential of a variety of transformed cell lines (see, e.g., Richon et al, 1996; Kim et al., 1999; Yoshida et al., 1995; Yoshida & Beppu, 1988). In vivo, phenybutyrate is reported to be effective in the treatment of acute promyelocytic leukemia in conjunction with retinoic acid (see, e.g., Warrell et al., 1998). SAHA is reported to be effective in preventing the formation of mammary tumours in rats, and lung tumours in mice (see, e.g., Desai et al., 1999).
The clear involvement of HDACs in the control of cell proliferation and differentiation suggests that aberrant HDAC activity may play a role in cancer. The most direct demonstration that deacetylases contribute to cancer development comes from the analysis of different acute promyelocytic leukemias (APL). In most APL patients, a translocation of chromosomes 15 and 17 (t(15;17)) results in the expression of a fusion protein containing the N-terminal portion of PML gene product linked to most of RARα (retinoic acid receptor). In some cases, a different translocation (t(11;17)) causes the fusion between the zinc finger protein PLZF and RARα. In the absence of ligand, the wild type RARα represses target genes by tethering HDAC repressor complexes to the promoter DNA. During normal hematopoiesis, retinoic acid (RA) binds RARα and displaces the repressor complex, allowing expression of genes implicated in myeloid differentiation. The RARα fusion proteins occurring in APL patients are no longer responsive to physiological levels of RA and they interfere with the expression of the RA-inducible genes that promote myeloid differentiation. This results in a clonal expansion of promyelocytic cells and development of leukaemia. In vitro experiments have shown that TSA is capable of restoring RA-responsiveness to the fusion RARα proteins and of allowing myeloid differentiation. These results establish a link between HDACs and oncogenesis and suggest that HDACs are potential targets for pharmaceutical intervention in APL patients. (See, for example, Kitamura et al., 2000; David et al., 1998; Lin et al., 1998).
Furthermore, different lines of evidence suggest that HDACs may be important therapeutic targets in other types of cancer. Cell lines derived from many different cancers (prostate, colorectal, breast, neuronal, hepatic) are induced to differentiate by HDAC inhibitors (Yoshida and Horinouchi, 1999). A number of HDAC inhibitors have been studied in animal models of cancer. They reduce tumour growth and prolong the lifespan of mice bearing different types of transplanted tumours, including melanoma, leukaemia, colon, lung and gastric carcinomas, etc. (Ueda et al., 1994; Kim et al., 1999).
Psoriasis is a common chronic disfiguring skin disease which is characterised by well-demarcated, red, hardened scaly plaques: these may be limited or widespread. The prevalence rate of psoriasis is approximately 2%, i.e., 12.5 million sufferers in the triad countries (US/Europe/Japan). While the disease is rarely fatal, it clearly has serious detrimental effects upon the quality of life of the patient: this is further compounded by the lack of effective therapies. Present treatments are either ineffective, cosmetically unacceptable, or possess undesired side effects. There is therefore a large unmet clinical need for effective and safe drugs for this condition.
Psoriasis is a disease of complex etiology. Whilst there is clearly a genetic component, with a number of gene loci being involved, there are also undefined environmental triggers. Whatever the ultimate cause of psoriasis, at the cellular level, it is characterised by local T-cell mediated inflammation, by keratinocyte hyperproliferation, and by localised angiogenesis. These are all processes in which histone deacetylases have been implicated (see, e.g., Saunders et al., 1999; Bernhard et al, 1999; Takahashi et al, 1996; Kim et al , 2001). Therefore HDAC inhibitors may be of use in therapy for psoriasis. Candidate drugs may be screened, for example, using proliferation assays with T-cells and/or keratinocytes.
Thus, one aim of the present invention Is the provision of compounds which are potent inhibitors of histone deacetylases (HDACs). There is a pressing need for such compounds, particularly for use as antiproliferatives, for example, anti-cancer agents, agents for the treatment of psoriasis, etc.
Such molecules desirably have one or more of the following properties and/or effects:                (a) easily gain access to and act upon tumour cells;        (b) down-regulate HDAC activity;        (c) inhibit the formation of HDAC complexes;        (d) inhibit the interactions of HDAC complexes;        (e) inhibit tumour cell proliferation;        (e) promote tumour cell apoptosis;        (f) inhibit tumour growth; and,        (g) complement the activity of traditional chemotherapeutic agents.        
A number of carbamic acid compounds have been described.
Certain classes of carbamic acid compounds which inhibit HDAC are described in Watkins et al., 2002a, 2002b, 2002c, 2003.
Quinolines
Kato et al., 1996, describe certain carbamic acid compounds bearing a quinolin-2-yl group (shown below) as potential agents for the treatment of neurodegenerative disorders.
Cmpd.PageStructureCAS No.1-341183965-19-7P 1-442183965-21-1P 1-542183965-22-2P 2-144183965-25-5P 2-244183965-26-6P 2-345183965-27-7P 2-445183965-28-8P 2-747183965-31-3P 2-847183965-32-4P 2-947183965-33-5P  2-1047183965-34-6P  2-1148183965-35-7P 1237186522-77-0P 2641186522-97-4P 2741186522-98-5P 2841186522-99-6P
Musser et al., 1988, describes carbamic acid compounds (including one bearing a quinolin-2-yl group, linked via a methylene-oxy-meta-phenylene ether group) as inhibitors of 5-lipoxygenase/cyclooxygenase and leukotriene antagonists for the treatment of inflammatory and allergic diseases.
CmpdPageStructureCAS No.48118308-98-8
Venkatesan et al., 2001, describe a carbamic acid compound bearing a quinolin-6-yl group (shown below) as an inhibitor of matrix metalloproteases and TNFα converting enzyme (TACE).
Cmpd.ColumnStructureCAS No.6750212766-63-7PBenzoxazoles and Related Compounds
Kato et al., 1996, describe a carbamic acid compound bearing a benzoxazol-2-yl group (shown below) as a potential agent for the treatment of neurodegenerauive disorders.
StructureCAS No.183963-81-7P
Turin et al., 1996, describe certain carbamic acids compounds bearing a furobenzoxazole group (shown below) as potential bronchodilators.
StructureCAS No.65874-35-3P 65874-38-6P
Fauran et al, 1974, describe a carbamic acid compound bearing a furobenzoxazole group (shown below) as a potential bronchodilator and hypotensive agent.
StructureCAS No.54414-41-4PBenzothiazoles
Baxter et al., 2000, describe a carbamic acid compound bearing a benzothiazol-2-yl group (shown below) as an inhibitor of matrix metalloproteases for use in therapy of a number of diseases, including cancer.
StructureCAS No.21344-60-5P
Kato et al., 1996, describe certain carbamic acid compounds bearing a benzothiazol-2-yl group (shown below) as agents for the treatment of neurodegenerative disease:
StructureCAS No.183963-77-1P 183963-85-1P
Babichev et al., 1968, describe the synthesis of certain carbamic acid compounds bearing a benzothiazol-2-yl group (shown below).
StructureCAS No.21344-60-5P 21344-61-6P 21344-62-7PBenzimidazoles
Strakov et al., 1972 describes the synthesis of a carbamic acid compound bearing a benzimidazol-2-yl group (shown below).
StructureCAS No.37454-68-5P
Guines et al., 1992 describes certain carbamic acids compound bearing a benzimidazol-2-yl group (shown below) that apparently have antifungal properties.
StructureCAS No.143949-73-9P 143949-76-2P